The unique CDP-choline pathway for phosphatidylcholine (PtdCho) biosynthesis present in spirochetes of the genus Treponema is likely both a consequence of and a contributor to the commensal/pathogenic lifestyles of these organisms, reflecting their coevolution with eukaryotic hosts. We hypothesize that the final enzyme step in T. denticola PtdCho synthesis (1,2- diacylglycerol choline phosphotransferase [CPT]) is encoded by TDE0021, a gene of apparent eukaryotic origin. In many eukaryotes, this enzyme also has 1,2-diacylglycerol ethanolamine phosphotransferase (EPT) activity required for phosphatidylethanolamine (PtdEtn) synthesis. In T. denticola, PtdEtn (synthesized by an as-yet unidentified pathway) is upregulated when the first step in PtdCho synthesis is blocked. We hypothesize that T. denticola TDE0021 encodes CPT activity and may also have EPT activity, thus making it the key enzyme in phospholipid synthesis and an attractive target for development of specific anti-Treponema agents against a range of mucosal and venereal treponematoses. This application addresses the following areas: Aim 1: To characterize the CPT activity of T. denticola required for synthesis of PtdCho. We will determine if T. denticola TDE0021 encodes CPT activity by (A) construction and characterization of an isogenic TDE0021 mutant and (B) complementation of a Saccharomyces cerevisiae CPT/EPT mutant with T. denticola TDE0021 DNA. Aim 2: To characterize synthesis and expression of PtdEtn in T. denticola. We will (A) identify and characterize the T. denticola PtdEtn synthesis pathway, and (B) characterize the mechanism(s) responsible for increased levels of PtdEtn in a defined PtdCho-deficient T. denticola mutant. Upon completion of the two Aims of this project, we will have made significant progress toward understanding the genetic components and mechanisms of the unique phospholipid synthesis pathway in the genus Treponema, which includes both mucosal and venereal pathogens. The knowledge gained will significantly enhance understanding of spirochete membrane biology, will provide a basis for continued studies of the role of the phospholipid environment on microbe-host interactions, and may provide the basis for development of novel therapeutic agents. PUBLIC HEALTH RELEVANCE: This project will characterize genetic components and mechanisms comprising the unique phospholipid synthesis pathways in the genus Treponema, which includes both mucosal and venereal pathogens. This will contribute to understanding of the role of the membrane phospholipid environment on microbe-host interactions, and may provide a basis for development of novel therapeutic agents.